Background/Rationale: Intestinal epithelial barrier integrity is critical for most gastrointestinal (GI) functions including digestion of food, as well as absorption and secretion of nutrients. The gastrointestinal mucosal layer is very vulnerable to injury resulting in loss of barrier integrity, and this occurs very commonly in critically ill Veteran patients in response to surgical stress. Preventing it would likely have a major impact on outcomes and costs for all critically ill Veteran populations, though the molecular controls that regulate barrier integrity are incompletely understood. Current treatment for barrier dysfunction is conservative. This application will explore a promising approach to preserve, protect, and restore epithelial barrier function by manipulating the fundamental regulation of intestinal epithelial cells; while improving our overall understanding of their function and regulation. The bioactive sphingolipid sphingosine-1-phosphate (S1P) has recently demonstrated significant effects on endothelial and epithelial cells in promoting important biological functions, such as survival of injured cells and expression of barrier proteins. We have demonstrated in previously published work and our preliminary data that S1P has significant effects on intestinal epithelial cells and the intestinal barrier; both in physiologic and pathologic conditions, though the details of this mechanistically have not been fully elucidated. Objectives: The primary objective of this study is to demonstrate that S1P protects the intestinal epithelial barrier in both physiological and pathological states with the eventual goal of having a therapeutic use for S1P in critically ill patients. Methods: This proposal includes in vitro studies using cultured intestinal epithelial cells, ex vivo studies of tissues from mice and humans, and in vivo studies in mice. Expected Results: We hypothesize that S1P will have significant effects as both an intracellular 2nd messenger and external receptor ligand to promote barrier integrity in cultured cells and in vivo, during physiological conditions and in pathological states. Status: Cells exposed to S1P show increased levels of barrier proteins and improved barrier function. Cells overexpressing Sphingosine Kinase 1, and expressing higher S1P content, show significantly increased proliferation and a decrease in microRNA (mir) 542 among other findings. Mice exposed to S1P show improved barrier function in control states and during ischemia- reperfusion, and also decreased levels of mir 542. MicroRNA 495 inhibits Sphingosine Kinase 1 and shows decreased proliferation and increased apoptosis. Impact: The proposed research addresses critical mechanisms in maintenance of an intact intestinal mucosal barrier, and in S1P, a potential therapy to preserve this function in critically ill patients. Critical illness, particularly in surgical patients, is very common and a high percentage of patients in the intensive care units incur barrier dysfunction. Despite the medical advances of the past few decades, therapies for these gastrointestinal injuries are, for the most part, conservative as there is no current pharmacologic therapy available. This proposal explores such a pharmacologic therapy that could potentially prevent injury and loss of the intestinal epithelial cells and lead to improved outcomes of critically ill Veteran patients.